A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; A Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification.

Keith E Belk,Enrique Doster,Lyndsey Linke,Pablo Rovira,Margaret D Weinroth,Xiang Yang,Christina Boucher,Noelle R Noyes,Roberta Magnuson,Brandy A Burgess,Paul S Morley

doi:10.3389/fmicb.2019.02499

Abstract

This study was conducted to compare aerobic culture, polymerase chain reaction (PCR), lateral flow immunoassay (LFI), and shotgun metagenomics for identification of Salmonella enterica in feces collected from feedlot cattle. Samples were analyzed in parallel using all four tests. Results from aerobic culture and PCR were 100% concordant and indicated low S. enterica prevalence (3/60 samples positive). Although low S. enterica prevalence restricted formal statistical comparisons, LFI and deep metagenomic sequencing results were discordant with these results. Specifically, metagenomic analysis using k-mer-based classification against the RefSeq database indicated that 11/60 of samples contained sequence reads that matched to the S. enterica genome and uniquely identified this species of bacteria within the sample. However, further examination revealed that plasmid sequences were often included with bacterial genomic sequence data submitted to NCBI, which can lead to incorrect taxonomic classification. To circumvent this classification problem, we separated all plasmid sequences included in bacterial RefSeq genomes and reassigned them to a unique taxon so that they would not be uniquely associated with specific bacterial species such as S. enterica. Using this revised database and taxonomic structure, we found that only 6/60 samples contained sequences specific for S. enterica, suggesting increased relative specificity. Reads identified as S. enterica in these six samples were further evaluated using BLAST and NCBI’s nr/nt database, which identified that only 2/60 samples contained reads exclusive to S. enterica chromosomal genomes. These two samples were culture- and PCR-negative, suggesting that even deep metagenomic sequencing suffers from lower sensitivity and specificity in comparison to more traditional pathogen detection methods. Additionally, no sample reads were taxonomically classified as S. enterica with two other metagenomic tools, Metagenomic Intra-species Diversity Analysis System (MIDAS) and Metagenomic Phylogenetic Analysis 2 (MetaPhlAn2). This study re-affirmed that the traditional techniques of aerobic culture and PCR provide similar results for S. enterica identification in cattle feces. On the other hand, metagenomic results are highly influenced by the classification method and reference database employed. These results highlight the nuances of computational detection of species-level sequences within short-read metagenomic sequence data, and emphasize the need for cautious interpretation of such results.

Highlights

The study and detection of microbial organisms has long been reliant on cultivation and characterization of certain species, but advancements in sequencing technologies have revealed an underlying microbial diversity largely ignored by culturebased techniques (Staley and Konopka, 1985; Hugenholtz, 2002; Stewart et al, 2018)
Utilizing samples obtained from another study of feedlot cattle (Doster et al, 2018), we compared a metagenomic approach for S. enterica identification to the traditional techniques of aerobic culture, polymerase chain reaction (PCR), and lateral flow immunoassays (LFI)
Aerobic culture and LFIs were used to test for the presence of S. enterica on 376 fecal samples collected from study cattle

Summary

Introduction

The study and detection of microbial organisms has long been reliant on cultivation and characterization of certain species, but advancements in sequencing technologies have revealed an underlying microbial diversity largely ignored by culturebased techniques (Staley and Konopka, 1985; Hugenholtz, 2002; Stewart et al, 2018). High-throughput sequencing techniques enable a culture-independent metagenomic approach that provides access to DNA from all bacteria (microbiome) within a given sample This rapidly developing technology provides great potential for investigating the complexity of bacterial communities (Turnbaugh et al, 2007; Forsberg et al, 2012; Noyes et al, 2016). Metagenomic approaches have been used to find novel pathogens when traditional methods were not fruitful (Gire et al, 2014; Zhou et al, 2016), but the relative increased sensitivity in metagenomic approaches raises important questions about their use in identification of foodborne pathogens in fecal samples One such example is Salmonella enterica, an important zoonotic pathogen that causes over 93 million cases of gastroenteritis in humans globally every year (Majowicz et al, 2010) and has been implicated in outbreaks associated with beef products (Laufer et al, 2015). Utilizing samples obtained from another study of feedlot cattle (Doster et al, 2018), we compared a metagenomic approach for S. enterica identification to the traditional techniques of aerobic culture, polymerase chain reaction (PCR), and lateral flow immunoassays (LFI)

Methods

Results

Conclusion